High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

Abstract

Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogues. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.

High‐Content Analysis in Toxicology: Screening Substances for Human Toxicity Potential, Elucidating Subcellular Mechanisms and In Vivo Use as Translational Safety Biomarkers

“…This is similar to that reported for regulatory animal toxicity studies [5] and more than double that of conventional cytotoxicity assays [12]. The basis for the lower concordance of this cytotoxicity assay with human toxicity than for HCA assays described by others [7,12,17,19,20,[22][23][24] is unknown. However, it may be attributable to a number of factors such as insufficient duration of drug exposure, use of non-proliferative cells or HepG2 cells were exposed for 3 days to various drugs at 12 different concentrations in duplicate rows stained with different cocktails of a total of 8 different dyes for detection of nine key parameters whose collective pattern is indicative of cytotoxicity mechanism.…”

“…Compelling support for the predictive potential of the quadprobe HCA cytotoxicity assay is provided by several studies comparing the relative toxicity of different drugs within the same chemical or pharmacological class [7,16,22,28], including the glitazones, statins, nucleoside reverse transcriptase inhibitors, quinalone antibiotics and antimicrobial peptides [24].…”

High-content analysis for drug delivery and nanoparticle applications

“…Images are acquired at 10 or 20 magnification and are subjected to segmentation analysis, yielding quantitative data for both individual cells and cell populations; these are used in tandem with the fluorescent micrographs taken during acquisition. Adapted, with permission, from [43]. Readings were compared with 100% negative controls.…”

“…Melittin not only perturbs bacterial membranes, but is also hemolytic to mammalian erythrocytes at similar concentrations [42]. HCA on Caco-2 cells examined systematic amino acid replacement in melittin analogs to discover whether peptides with the best efficacy and lowest effect on cell cytotoxic parameters could be synthesized [43]. Reduced changes in plasma membrane potential and mitochondrial membrane potential were seen with analogs with lower hydrophobicity compared with the parent melittin molecule, but unfortunately this also led to a reduction in transcellular permeation enhancement.…”

High content analysis: A sensitive tool to detect and quantify the cytotoxic, synergistic and antagonistic effects of chemical contaminants in foods

“…HCA has been shown to be highly sensitive and specific using pre-lethal toxicity assays to establish subtle changes in cell health rather than overt, gross cytotoxicity detected by conventional 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) and neutral red (NR) assays O'Brien and Haskins, 2007;O'Brien, 2008O'Brien, 2008Taylor, 2007;Xu et al, 2004). It is capable of combining automated fluorescence microscopy and advanced image analysis software, to measure multiple cellular events in single cells (Walsh et al, 2011).…”